3′-exonuclease, production and use thereof

ABSTRACT

The present invention relates to a poly(A)-specific 3′-exonuclease activity which can be obtained by chromatographically purifying a crude extract of animal or human cells and to its use for deadenylating 3′-poly(A) tails of nucleic acids and as a pharmaceutical or diagnostic agent, or for identifying functional interactors.

DESCRIPTION

The present invention relates to a poly(A)-specific 3′-exonucleaseactivity which can be obtained by chromatographically purifying a crudeextract of animal or human cells and to its use for deadenylating3′-poly(A) tails of nucleic acids and as a pharmaceutical or diagnosticagent, or for identifying functional interactors.

Most eukaryotic mRNAs carry poly(A) tails of approx. 200 adenosineresidues in length at their 3′ ends. These poly(A) tails appear toinfluence not only the half-life or intracellular transport of mRNAs butalso translation of the mRNA into the corresponding protein. While theprecise mechanism has still not been elucidated, the synthesis anddegradation of poly(A) tails appear to be directly or indirectlyconnected with the function of the tails (see, e.g., Wickens, M. et al.(1977) Curr. Opin. Genet. Dev., 7, 220-232).

Poly(A)-degrading nuclease activities have already been investigated inseveral eukaryotic systems (see, e.g., Virtanen, A. & Åström, J. (1997)in Prog. Mol. Subcell. Biol. (Jeanteur, P., ed.) Vol. 16, 199-220,Springer-Verlag, Berlin-Heidelberg). Thus, two reaction pathways have,for example, been identified in yeasts. One of the reaction pathways,i.e. the so-called deadenylation dependent decapping passway, is startedby removal of the poly(A) tail and concerns a 5′-3′-exonucleolyticdegradation of mRNA by an Xm1p5′-exonuclease. The other reactionpathway, i.e. the so-called 3′-5′-decay passway, is started by adeadenylation of the mRNA and concerns a 3′-5′-exoribonucleolyticdegradation of mRNA. A multicomponent complex, termed an exosome, hasrecently been identified as being involved in the 3′-5′-decay passway(Mitchell, P. et al. (1997), Cell 91, 457-466). The exosome consists ofseveral 3′-5′-exoribonucleases and is involved both in 5.8S rRNA 3′processing and in the 3′-5′ degradation of mRNA (Anderson, J. S. J. &Parker, R. (1998) EMBO J. 17, 1497-1506). However, it is not knownwhether any of the exoribonucleases of the exosome degrade poly(A)preferentially. In addition to this, a poly(A)-binding protein I(PABI)-dependent poly(A)-specific nuclease (PAN) has been identified inyeasts (see, e.g., Lowell, J. E. et al. (1992) Genes Dev. 6, 2088-2099).PAN is a 3′-5′-exoribonuclease and is composed of at least twopolypeptides, i.e. Pan2p and Pan3p (see, e.g., Brown, C. E. Jr. et al.(1996) Mol. Cell. Biol., 16, 5744-5753).

At least three different poly(A)-degrading activities have beencharacterized in mammalian cells. For example, an activity found in Helacells has a high selectivity for degrading 3′-located poly(A) tails,requires 3′-located hydroxyl groups and forms 5′-AMP as themononucleotide reaction product (see, e.g., Åström, J. et al. (1992) J.Biol. Chem. 267 (25), 18154-18159 and J. Aström, A. Aström and A.Virtanen, In vitro deadenylation of mammalian mRNA by a HeLa cell 3′exonuclease, EMBO J., (1991), Vol 10, 3067). Furthermore, anMg²⁺-dependent poly(A)-specific 3′-exoribonuclease having a molecularweight of 74 kDa (Körner, C. G. & Wahle, E. (1997) J. Biol. Chem., 272(16), 10448-10456) has been described in calf thymus. Apolyribosome-associated 3′-exoribonuclease having a molecular weight of33 kDa has also been described, but this 3′-exoribonuclease is notspecific for poly(A) (Caruccio, N. & Ross, J. (1994) J. Biol. Chem. 269(50), 31814-31821). Another poly(A)-specific 3′-exonuclease activityhaving a molecular weight of 60 kDa was identified in calf thymus.However, it subsequently turned out that this protein is the hnRNP Lprotein and consequently has no connection with the poly(A)-specific3′-exoribonuclease activity which has been measured.

The object of the present invention was therefore to make available a3′-exoribonuclease which specifically degrades 3′-located poly(A) tails.

The present invention therefore relates to a process for isolating apoly(A)-specific 3′-exonuclease activity, which process contains thefollowing steps:

a) preparing a crude extract from animal or human cells;

b) precipitating the protein present in the crude extract obtainablefrom step (a);

c) subjecting the precipitate obtainable from step (b) to chromatographyon a basic anion exchanger;

d) subjecting the active fractions from step (c) to affinitychromatography;

e) subjecting the active fractions from step (d) to chromatography on abasic anion exchanger;

f) subjecting the active fractions from step (e) to affinitychromatography;

g) subjecting the active fractions from step (f) to poly(A)-affinitychromatography;

h) subjecting the active fractions from step (g) to chromatography on abasic anion exchanger; and, where appropriate,

i) subjecting the active fractions from step (g) to gel filtration. Or

j) subjecting the active fractions from step (f) to two rounds ofaffinity chromatography;

Surprisingly, the 3′-exonuclease activity which can be obtained by theabove-described process is specific for 3′-located poly(A) tails, with a3′-located hydroxyl group being required and with 5′-AMP being formed asthe mononucleotide reaction product. In contrast to the already known3′-exonuclease activities, the 3′-exonuclease activity according to theinvention has a molecular weight of approx. 50 kDa under denaturingconditions. In contrast to the 74 kDa protein from calf thymus, the3′-exonuclease activity according to the invention is not stimulated byspermidine at low salt concentrations; on the contrary, if anything, itis inhibited both at low and at high salt concentrations. Furthermore,in contrast to the calf thymus 74 kDa protein, the 3′-exonucleaseaccording to the invention interacts relatively strongly with HeparinSepharose. Furthermore, the calf thymus 74 kDa protein is not found inthe SDS-Page—FIGS. 1B and 2B. In addition, it was surprising that, incontrast to the HeLa cell exonuclease activity, the exonucleaseaccording to the invention is also active in the presence of Mn²⁺. It isalso surprising that the exonuclease according to the invention operatesprogressively, i.e. the exonuclease binds to the 3′ end of the poly(A)tail and degrades it nucleotide by nucleotide without theexonuclease-poly(A) complex dissociating, whereas the 74 kDa proteinoperates distributively, with the complex dissociating after oneoperational step and having to be regenerated.

The poly(A)-specific 3′-exonuclease activity according to the inventioncan preferably be isolated from animal or human thymus cells, inparticular from calf thymus. In general, a whole cell extract isprepared for this purpose, with protein preferably being precipitatedfrom this extract with ammonium sulfate. In this connection, preferenceis given to the saturation concentration being approx. 45% ammoniumsulfate. It has been found to be particularly advantageous in thisconnection if, before the true protein precipitation, foreign proteinsare separated off by being precipitated at a saturation concentration ofpreferably approx. 25% ammonium sulfate, such that the desired3′-exonuclease activity can, in a subsequent step, be precipitated outof the supernatant at an ammonium sulfate saturation of approx. 45%.This protein fractionation itself separates off a considerable portionof unwanted foreign proteins.

The precipitate is then subjected to chromatography on a basic anionexchanger, preferably on a weakly basic anion exchanger, in particularon DEAE, such as DEAE-Sepharose. In general, the active fraction elutesat an approx. 0.17 M concentration of a salt, preferably a monovalentsalt such as KCl. The eluted active fraction, which has been dialyzed ina customary manner, is then subjected to an affinity chromatography,preferably on heparin, since it has been found, surprisingly, that theactive fraction binds particularly well to heparin, e.g. HeparinSepharose. The active fractions are therefore usually eluted at highsalt concentrations, for example at an approx. 1.0 M concentration of amonovalent salt such as KCl. The active fractions are then subjected tochromatography on a basic anion exchanger, preferably on a stronglybasic anion exchanger, in particular on Mono Q, such as Mono Q HR 16/10.The proteins are preferably eluted by means of a linear gradient, withit being possible to elute the active fractions at an approx. 10%concentration of a salt, in particular a monovalent salt such as KCl,whose concentration is approx. 1.0 M. After that, the active fractionsare subjected to affinity chromatography, preferably on a dye, inparticular on a blue dye, very particularly on Blue Sepharose.Preference is given to eluting the proteins using a multistep, inparticular a two-step, salt gradient, with the active fractionspreferably eluting well at a high salt concentration, in particular athigh concentrations of a monovalent salt, very particularly at anapprox. 1.0 M concentration, such as 1.0 M KCl.

According to the present invention, this is then followed by an affinitychromatography on poly(A), with it being possible to elute the activefractions at an approx. 0.35 to approx. 0.55 M concentration of a salt,in particular a monovalent salt, such as KCl. In addition, in conformitywith the process according to the invention, the active fractions aresubjected to chromatography on another basic anion exchanger, preferablyon a strongly basic anion exchanger, in particular on Mono Q, such asSMART Mono Q. In this connection, the activity is preferably eluted atan approx. 0.17 M concentration of a salt, in particular a monovalentsalt such as KCl. In conformity with the process according to theinvention, the last purification step is, where appropriate, a step inwhich the active fractions are subjected to a gel filtration, preferablya Superdex 200 gel filtration, in particular a SMART Superdex 200 gelfiltration, with it generally being possible to fractionate the activefractions satisfactorily in the presence of an approx. 0.1 Mconcentration of a salt, in particular a monovalent salt such as KCl.

In conformity with the process according to the invention, it ispossible to purify a poly(A)-specific 3′-exonuclease activity approx.600-fold with a yield of approx. 13% (see Table I). In this connection,it was particularly surprising that the poly(A) affinity chromatographyin accordance with step (g) resulted in an approx. 14-fold purificationof the activity according to the invention.

In another embodiment, the proteins are subjected, after the dyechromatography, to a double affinity chromatography, preferablychromatography on ssDNA Agarose followed by chromatography on 5′AMPSepharose. The active fractions are normally eluted at high saltconcentrations, such as an approx. 2.0 M concentration of a monovalentsalt such as KCl.

The process according to the invention now results in the isolation of apoly(A)-specific 3′-exonuclease activity which runs at approx. 50 kDaunder denaturing conditions, for example in an SDS-PAGE gel, and runs atfrom approx. 180 to 220 kDa under native conditions, for example onSuperdex 200.

The invention therefore also relates to an approx. 50 kDa protein and,where appropriate, an associated protein (tetramer) which possesses apoly(A)-specific exonuclease activity and which can be obtained inaccordance with the process according to the invention.

The present invention therefore also relates to a composition whichcomprises a poly(A)-specific 3′-exonuclease activity which can beobtained in accordance with the process according to the invention.Where appropriate, the composition comprises other additives andadjuvants.

The present invention also relates to a process for deadenylatingnucleic acids, in particular for deadenylating 3′-located poly(A) tailsbelonging, preferably, to mRNA in the presence of a compositionaccording to the invention. The deadenylation reaction preferably takesplace in the presence of monovalent cations, such as K⁺ and/or Na⁺, inparticular at concentrations of the monovalent cation of approx. 0.1 M.Preference is furthermore given to the deadenylation reaction takingplace at a pH of approx. 7.

The present invention also relates to antibodies which reactspecifically with the composition according to the invention and/or acomponent thereof, with the composition itself being immunogenic or withit being possible to make the composition immunogenic, or to increasethe immunogenicity of the composition, by coupling it to suitablecarriers such as bovine serum albumin.

The antibodies are either polyclonal antibodies or monoclonalantibodies. Their preparation, which is also part of the subject matterof the present invention, is effected, for example in accordance withwell-known methods, by immunizing a mammal, for example a rabbit, withthe composition according to the invention, where appropriate in thepresence of, for example, Freund's adjuvant and/or aluminum hydroxidegels (see, e.g., Diamond, B. A. et al. (1981) The New England Journal ofMedicine, 1344). The polyclonal antibodies which are produced in theanimal due to an immunological reaction can then readily be isolatedfrom the blood in accordance with well-known methods and, for example,purified by means of column chromatography. Preference is given topurifying the antibodies by affinity chromatography, in which, forexample, the composition according to the invention has been coupled toan NHS-activated HiTrap column.

Monoclonal antibodies can, for example, be prepared in accordance withthe known method of Winter & Milstein (Winter, G. & Milstein, C. (1991)Nature, 349, 293).

The present invention furthermore also relates to a pharmaceutical whichcomprises a composition according to the invention and, whereappropriate, suitable additives or adjuvants and to a process forproducing a pharmaceutical for treating cancer, autoimmune diseases, inparticular multiple sclerosis or rheumatoid arthritis, Alzheimer'sdisease, allergies, in particular neurodermatitis, type I allergies ortype IV allergies, arthrosis, atherosclerosis, osteoporosis, acute andchronic infectious diseases and/or diabetis, and/or for influencing themetabolism of the cell, in particular in association withimmunosuppression, very particularly in association withtransplantations, in which pharmaceutical a composition according to theinvention is formulated together with pharmaceutically acceptableadditives and/or adjuvants.

Examples of suitable additives and/or adjuvants are a physiologicalsodium chloride solution, stabilizers, proteinase inhibitors, etc.

The present invention furthermore also relates to a diagnostic agentwhich comprises a composition according to the invention and, whereappropriate, suitable additives and/or adjuvants and to a process forpreparing a diagnostic agent for diagnosing cancer, autoimmune diseases,in particular multiple sclerosis or rheumatoid arthritis, Alzheimer'sdisease, allergies, in particular neurodermatitis, type I allergies ortype IV allergies, arthrosis, atherosclerosis, osteoporosis, acute andchronic infectious diseases and/or diabetis, and/or for analyzing themetabolism of the cell, in particular the immune status, veryparticularly in association with transplantations, in whichpharmaceutical suitable additives and/or adjuvants are added to acomposition according to the invention.

For example, according to the present invention, the compositionaccording to the invention can be bound to a solid phase, e.g.consisting of nitrocellulose or nylon, and in this way be brought intocontact in vitro, for example, with the body fluid to be investigated,e.g. blood, in order thereby to be able to react, for example, withautoimmune antibodies. The antibody-peptide complex can then, forexample, be detected using labeled antihuman IgG or antihuman IgMantibodies. The label is, for example, an enzyme, such as peroxidase,which catalyzes a color reaction. The presence of autoimmune antibodies,and the quantity of the antibodies which is present, can thereby bedetermined readily and rapidly by way of the color reaction.

Another diagnostic agent comprises the antibodies according to theinvention themselves. Using these antibodies it is possible, forexample, to readily and rapidly investigate a human tissue sample todetermine whether the composition according to the invention and/or acomponent thereof is present. In this case, the antibodies according tothe invention are labeled, for example, with an enzyme as has alreadybeen described above. This enables the specific antibody-peptide complexto be determined readily and just as rapidly by way of an enzymic colorreaction.

The present invention also relates to a test for identifying functionalinteractors, such as inhibitors or stimulators, comprising a compositionaccording to the invention or antibodies according to the invention and,where appropriate, suitable additives and/or adjuvants. For this,selected substances, for example from a so-called chemical library, areemployed in the deadenylation reaction which has already been describedin detail above and the activity of the composition according to theinvention is measured in the presence and/or absence of the substances.An example of a suitable substrate is mRNA or poly(A). The test can becarried out, for example, in analogy with the in-vitro deadenylation, asdescribed in detail in the examples.

Another general possibility of using the composition according to theinvention is therefore also that of degrading nucleic acids, inparticular mRNA, in a poly(A)-specific manner. The poly(A)-specificdegradation of nucleic acids can be of particular use in researchlaboratories.

The following tables, figures and examples are intended to clarify theinvention without limiting it.

DESCRIPTION OF THE TABLES AND FIGURES

Table 1 summarizes the purification of bovine poly(A)-specific3′-exonuclease activity. The deadenylation activity was quantified byincubating the chromatographic fractions, under conditions for in-vitrodeadenylation, with L3(A₃₀) RNA substrate which had been labeled with[α³²P]ATP during in-vitro transcription. One unit is defined as therelease of 1 pmol of AMP per minute.

TABLE 1 Protein Activity Specific activity Purification Fraction mgUnits × 10⁻³ Units × 10⁻³/mg fold A.S.45 65,280 72,000 1.1 — II 3360100,000 29.9 27 IIB 652 59,000 90 82 MQ 43.2 17,000 387 352 C 14.4 9,000654 595

Table 2 shows the substrate specificity of the poly(A)-specific3′-exonuclease activity.

TABLE II Substrate Km(M) Rel. Vmax/Km poly(A) 1 × 10⁻⁸ 1 poly(U) 2 ×10⁻⁸ 1/10 poly(C) 1 × 10⁻⁸ 1/110 poly(G) 7 × 10⁻⁹ 1/240

FIG. 1A shows the identification of the 50 kDa polypeptide by means ofSMART Mono Q chromatography. In this connection, the Poly(A) Sepharosefraction was fractionated by SMART Mono Q chromatography and theresulting fractions were incubated, for 90 minutes, with uniformlylabeled RNA substrate L3 (A₃₀) under in-vitro deadenylation conditions.The reaction products were fractionated by electrophoresis in a 10%polyacrylamide:bisacrylamide 19:1-7 M urea gel. The resulting fluorogramis depicted in FIG. 1A. Lane S denotes RNA substrate which is incubatedin the absence of a fraction. Lane “Load MQ” denotes RNA substrate whichis incubated with a Poly(A) Sepharose fraction. Lanes 6 to 18 denote RNAsubstrate which is incubated in the presence of the fractions which wereobtained. Only even-numbered fractions were labeled.

FIG. 1B shows an SDS-PAGE of the SMART Mono Q fractions. In this case,aliquots of the fractions were separated by SDS-PAGE and the resultinggel was stained with silver. The fraction numbers are shown. Only evenfractions were labeled. The molecular weight markers were separated inlane M. The numbers on the left-hand side indicate the molecular weightsof the marker proteins in kDa.

FIG. 2A shows the identification of the 50 kDa polypeptide by means ofSMART Superdex 200 chromatography. The resulting fractions wereincubated, for 120 minutes, with uniformly labeled RNA substrate L3(A₃₀) under in-vitro deadenylation conditions. The reaction productswere fractionated on a 10% polyacrylamide:bisacrylamide 19:1-7 M ureagel. The resulting fluorogram is depicted in FIG. 2A. The L3 (A₃₀) laneshows the RNA substrate when incubated in the absence of a fraction. TheLoad lane shows the RNA substrate when incubated with MonoQ-concentrated Poly(A) Sepharose fraction. Lanes 15 to 25 show RNAsubstrate incubated in the presence of the resulting fractions. S and Pindicate the migration sites of RNA substrate (S) and products (P). Theelution profile of the molecular weight markers for calibrating theSuperdex 200 column is depicted at the top.

FIG. 2B shows an SDS-PAGE of SMART Superdex 200 fractions. The resultinggel was stained with silver. The fraction numbers are shown. Only evenfractions were labeled. The molecular weight markers were separated inlane M. The numbers on the left-hand side indicate the molecular weightsof the marker proteins in kDa.

FIG. 2C shows the 5′AMP Sepharose 4B affinity fractionation of specificpoly(A) exonuclease activity. Labeled fractions were incubated for 5 minwith radioactive L3 (A30) RNA substrate. The reaction products werefractionated on a 10% polyacrylamide:bisacrylamide 19:1-7 M urea gel.The resulting fluorogram is depicted in FIG. 2C. In lane A, RNAsubstrate was incubated with 5′AMP Sepharose 4B affinity-purifiedexonuclease. In lane “-”, the incubation was only with buffer in theabsence of the active fraction.

FIG. 2D shows an SDS-PAGE of 5′-AMP affinity chromatography on Sepharose4B. The resulting gel was stained with silver. The fraction numbers areindicated. Only even fractions were labeled. The molecular weightmarkers were separated in lane M. The numbers on the left-hand sideindicate the molecular weights of the marker proteins in kDa.

FIG. 3 shows the specificity of the exonuclease for 3′ end-locatedpoly(A) tails. The Poly(A) Sepharose fractions were incubated, underin-vitro deadenylation conditions, with the RNA substrates L3 (A₃₀), L3(A₃₀)X₁₅, L3(A₃₀)X₄₉ and L3(A₃₀)X₁₆₄, as indicated, for 0, 5, 10, 20,30, 60 or 90 minutes, as indicated. The reaction products werefractionated in a 10% polyacrylamide:bisacrylamide 19:1-7 molar ureagel. The resulting fluorogram is shown in FIG. 3. S and P indicate themigration sites of the RNA substrates (S) and products (P).

FIG. 4 shows the 5′-AMP reaction product which is released during thedeadenylation. The Poly(A) Sepharose fraction was incubated, for 20minutes, under in-vitro deadenylation conditions, with L3(A₃₀) RNAsubstrate which had been labeled with [α-³²P]ATP during in-vitrotranscription. 3 μl of the reaction mixture were subjected to a 2-D TLC.The resulting autoradiogram on the dried PEI cellulose plate is shown inFIG. 4. The migration sites of 2′-AMP, 3′-AMP and 5′-AMP markers, whichwere fractionated together with the radioactive sample, are indicated.

FIGS. 5A-C show the ongoing degradation of poly(A). The Poly(A)Sepharose fraction was incubated, under in-vitro deadenylationconditions, with X fmol of the L3(A₃₀) RNA substrate which had beenlabeled with [α-³²P]UTP during in-vitro transcription. The reactionproducts were fractionated in a 10% polyacrylamide:bisacrylamide 19:1-7M urea gel. The resulting fluorogram is shown in FIGS. 5A-C. S and Pindicate the migration sites of the RNA substrate (S) and the product(P). In this connection, FIG. 5A shows reactions which were carried outin the presence of 0.5 μl Poly(A) Sepharose fractions and terminated atthe times indicated. FIG. 5B shows reactions which were carried out inthe presence of 0.5 μl Poly(A) Sepharose fractions and terminated after20 minutes. The indicated quantities of poly(A) in ng were added to thereactions. FIG. 5C shows reactions which were carried out in thepresence of the indicated quantities of Poly(A) Sepharose fraction in μand terminated after 20 minutes.

FIG. 6A:

SDS-PAGE for the refolding experiment in accordance with Dayle A. Hagerand Richard R. Burgess, Anal. Biochem. (1980), 109, 76. The resultinggel was stained with silver. The numbers on the left-hand side indicatethe molecular weights of the marker proteins in kDa. The resulting bandswere excised, as indicated in FIG. 6A (A, 110, B, C, D and E).

FIG. 6B:

Denaturing RNA polyacrylamide gel: The gel pieces obtained in FIG. 6Awere eluted as described by Hager et al. The eluted proteins which wereobtained were incubated with the L3(A₃₀) substrate in accordance withEx. 9 and the reaction mixtures were fractionated on a polyacrylamidegel. The activity was demonstrated to be present in gel piece C (approx.50 kDa).

FIG. 7 shows the chromatographic steps involved in the process accordingto the invention.

ABBREVIATIONS USED IN THE TEXT

L3: Poly(A) site in the late region of human adenovirus 2 (length: 54nucleotides (J. Aström, A. Aström and A. Virtanen, In vitrodeadenylation of mammalian mRNA by a HeLa cell 3′ exonulease, EMBO,(1991), Vol 10, 3067)

X_(n): n nucleotides (A,G,T,C)

EXAMPLES Example 1

Preparing Cell-free Extracts

Crude whole-cell extracts were prepared from calf thymus in the mannerdescribed by Wahle, E. (1991) J. Biol. Chem. 266, 3131-3139. For this,from 3 to 15 kg of frozen calf thymus were thawed on ice, cut intopieces and homogenized, in an approximately equal quantity by volume ofbuffer 1 (50 mM Tris-HCl, 10 mM K₃PO₄, 1 mM EDTA, 10% glycerol, 50 mMKCl, 0.1 mM DTT, pH 7.9), in a Waring blender for 50 seconds at lowspeed and 50 seconds at high speed. The solid material was precipitatedby centrifuging in a Sorwall GSA rotor at 16,000 g and 4° C. for 60minutes. The crude extract was then filtered through a sieve having amesh aperture of 7 (normal) (Pascal Eng. Co. Ltd.); 0.134 g of ammoniumsulfate/ml (25% saturation) was then added and the extract was stirredon ice for 2 hours and the precipitate then precipitated by centrifugingin a Sorwall GSA rotor at 16,000 g and 4° C. for 60 minutes. A further0.115 g of ammonium sulfate was then added per ml of supernatant (45%saturation) and the supernatant was then treated as already describedabove. The pellet which was obtained after the centrifugation wasdissolved in from 2 to 4 volumes of buffer D (20 mM HEPES (KOH), 100 mMKCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol, pH 8.2) andthis solution was dialyzed at 4° C. for 10 hours in a dialysis baghaving a molecular weight exclusion limit of from 6000 to 8000. Afterthe dialysis, the 45% ammonium sulfate fraction (A.S. 45) was frozen inliquid nitrogen and stored at −70° C. When 4.5 kg of calf thymus wasused as the starting material, the A.S. 45 fraction (approx. 960 ml)then contained approx. 68 mg of protein per ml, giving a total quantityof protein of 65 g. The protein concentration was determined with aBiorad protein assay kit (No. 500-0001) using bovine gammaglobulin asthe reference substance.

Example 2

Partially Purifying a Poly(A)-specific 3′-exonuclease Activity

960 ml of crude A.S. 45 fraction (65 g of protein) were used as thestarting material for the partial purification (see Table I). Twoessentially identical DEAE Sepharose chromatographies were carried out.In the first, 160 ml of the A.S. 45 extract were added to DEAE SepharoseCL-6B (Pharmacia No. 17-0710-01) ion exchanger (240 ml of matrix in theform of spheres) which had been equilibrated with buffer D. Thesuspension was stirred slowly at 4° C. for 30 minutes. Unbound materialwas removed by washing three times with buffer D and then centrifuging(Sorwall H 4000 rotor at 800 rpm for 3 minutes). The DEAE Sepharosematrix was then packed into a column (diameter 50 mm) and washed withbuffer D at a flow rate of 9 cm per hour. A two-step salt gradient(buffer D containing 0.17 M and 1.0 M KCl, respectively) was carried outand the eluted protein was collected. The other 800 ml of the A.S. 45fraction were fractionated in the same way. However, in this case, 240ml of packed DEAE Sepharose were used and the column diameter was 70 mm.The protein which eluted at 0.17 M KCl, termed fraction II, was dialyzedagainst buffer D for 10 hours, frozen in liquid nitrogen and stored at−70° C. Subsequently, fraction II was fractionated by chromatography onHeparin Sepharose Cl-6B (Pharmacia No. 17-0467-01). The column, having abed volume of 100 ml and a diameter of 50 mm, was equilibrated withbuffer D at a flow rate of 9 cm per hour. Two essentially identicalHeparin Sepharose fractionations were carried out. Fraction II (210 ml)was loaded onto a Heparin Sepharose Cl-6B column having a flow rate of 9cm per hour. After the non-binding material had been eluted, boundprotein was eluted by washing the column with buffer D containing 1.0 MKCl. The Heparin Sepharose column was used a second time in order toseparate the remainder of fraction II (270 ml). The eluted fractions(termed IIB) were dialyzed, at 4° C., for 10 hours against buffer D,frozen in liquid nitrogen and stored at −70° C. Fraction IIB was thenfractionated by means of four essentially identical FPLC (Pharmacia)chromatographies on Mono Q HR 16/10 (Pharmacia No. 17-0506-01). Forthis, a Mono Q column which had been equilibrated with buffer D wasloaded with protein using a 50 ml Superloop (Pharmacia No. 19-7850-01)(approx. 30 ml of fraction IIB per cycle). The protein was eluted usinga two-step linear gradient (0 to 20% 1.0 M KCl in 250 ml of buffer D and20 to 50% 1.0 M KCl in 150 ml of buffer D), with 9 ml fractions beingcollected and the active fractions, which eluted at 10% 1.0 M KCl, beingcombined (termed fraction MQ), dialyzed at 4° C. for 10 hours againstbuffer D, frozen in liquid nitrogen and stored at −70° C. A 21 ml columnpacked with Blue Sepharose CL-6B (Pharmacia No. 17-0830-01) and having adiameter of 26 mm was prepared in accordance with the manufacturer'sinstructions. The column was equilibrated with buffer D at a flow rateof 34 cm per hour. The MQ fraction (108 ml) was loaded onto the columnand bound protein was eluted using a two-step salt gradient (buffer Dcontaining 0.17 M and 1.0 M KCl, respectively). The active fraction,termed C, which was obtained by eluting with 1.0 M KCl, was dialyzed at4° C. for 10 hours against buffer D, frozen in liquid nitrogen andstored at −70° C.

Example 3

Poly(A) Sepharose Chromatography

Poly(A) Sepharose CL-6B was prepared in accordance with themanufacturer's instructions. An HR 10/10 column having a bed volume of 8ml was equilibrated with buffer D containing 25 mM KCl at pH 7.12 mg (12ml) of fraction C were dialyzed for 4 hours against 2×2 l of buffer Dcontaining 25 mM KCl at pH 7. The dialyzed fraction was loaded onto thecolumn having a flow rate of 1 ml per minute. The column was thenfirstly washed with 5 bed volumes of buffer D containing 25 mM KCl at pH7 and, after that, washed with 5 bed volumes of buffer D containing 200mM KCl at pH 6 and, subsequently, with 5 bed volumes of buffer Dcontaining 280 mM KCl at pH 6. A gradient (5 bed volumes) of from 280 to600 mM KCl at pH 6 was then applied. The poly(A)-specific exonucleaseactivity eluted between 350 and 550 mM. This achieved a 14-foldpurification of the nuclease activity.

Example 4

SMART Mono Q Chromatography

A SMART Mono Q PC 1.6/5 column was equilibrated with buffer D containing50 mM KCl at pH 7 at a flow rate of 50 μl per minute. 1 ml of thePoly(A) Sepharose fraction (60 μg of protein) was dialyzed againstbuffer D containing 50 mM KCl at pH 7 and applied to the column at thesame flow rate. The column was then loaded with a 1.5 ml gradient offrom 50 to 500 mM KCl. 50 μl fractions were collected and theexonuclease activity was identified using an in-vitro deadenylation test(see below). The exonuclease activity eluted at approx. 170 mM KCl.

Example 5

SMART (Pharmacia, Uppsala) Superdex 200 Gel Filtration

The Poly(A) Sepharose fraction (0.7 ml, 0.06 mg per ml of protein) wasfirstly concentrated by means of a SMART Mono Q PC 1.6/5 (Pharmacia No.17-0671-01) chromatography which was carried out in accordance with thefollowing method. The column was equilibrated with buffer D containing50 mM KCl at pH 7. The Poly(A) Sepharose fraction was dialyzed againstbuffer D containing 50 mM KCl at pH 7 and loaded onto a Mono Q columnhaving a flow rate of 50 μl per minute. Bound material was eluted with alinear gradient of up to 500 mM KCl and 25 μl fractions were collected.The active fractions (total volume 100 μl) were identified and combined.50 μl of the concentrated Poly(A) Sepharose fraction were thenfractionated by gel filtration on a SMART Superdex 200 PC 3.2/30 columnwhich had been equilibrated with buffer D containing 100 mM KCl at pH 7.The flow rate was 40 μl per minute. The active fractions were identifiedusing an in-vitro deadenylation test (see below). The molecular weightmarkers were fractionated on a Superdex 200 column using the samemethod. The molecular weight markers were ferritin, catalase, aldolaseand BSA having molecular weights of 440, 232, 158 and 67 kDa,respectively.

Example 6

ssDNA Agarose Affinity Chromatography

ssDNA Agarose was prepared in accordance with the manufacturer'sinstructions (Pharmacia 27-5575-02). A 0.6 ml column was packed. Thecolumn was loaded with the active fraction C (10 ml) from Example 3 inbuffer D containing 50 ml of KCl (pH 7.0). The run-through fraction wascollected.

Example 7

5′-AMP Affinity Chromatography

5-AMP Sepharose (Pharmacia 17-0620-01) was prepared in accordance withthe manufacturer's instructions and the run-through fraction fromExample 6 was loaded onto the column in buffer D containing 50 ml of KCl(pH 7.0). The column was then washed with 5 ml of buffer D in 50 ml ofKCl (pH 7.0). After that, it was washed with 5 ml of buffer D containing200 ml of mM KCl (pH 7.0). Finally, the activity was eluted from thecolumn with 2 ml of buffer D containing 2 M KCl (pH 7.0).

Example 8

SDS-polyacrylamide Gel Electrophoresis (Carried Out for FIGS. 1B, 2B and2D)

SDS-polyacrylamide (acrylamide:bisacrylamide 30:0.8) gels (5% acrylamidein the spacer and 7.5% acrylamide in the resolving gel) were prepared asdescribed by Laemmli, U.K. (1970) Nature 227, 680-685 using aMini-protean II gel appliance (Bio-Rad No. 125BR). The appropriatequantities of protein were either fractionated directly on the gel or,before loading the gel, precipitated by adding 1 volume of 20% TCAfollowed by a centrifugation at 13,000 g and 4° C. for 30 minutes. Theresulting precipitate was washed with acetone, dissolved in 10 μl ofsample buffer (50 mM tris-HCl, pH 6.8, 1% (w/v) SDS, 100 mM DTT, 8%(v/v) glycerol, 0.025% (w/v) bromophenol blue) and fractionated by gelelectrophoresis. The resulting gel was fixed and stained. The silverstaining was performed as described by Oakley, B. R. et al. (1980) Anal.Biochem. 105, 361-363. The Commassie Brilliant Blue staining was carriedout as described by Merryl, C. R. (1990) Meth. Enzymol. 182, 477-488.

Example 9

Preparing RNA Substrates

The 5′-capped RNA substrates (L3(A₃₀), L3(A₃₀)X₁₅, L3(A₃₀)X₄₉ andL3(A₃₀)X₁₆₄) were synthesized by in-vitro transcription using T3 RNApolymerase (Promega No. P208C) and the plasmid pT3L3(A₃₀) (Åström, J. etal. (1991) EMBO J. 10(10), 3067-3071) as the DNA template, which wasdigested with NsiI, Hinc II, Eco RI and Pvu II. The RNA substrates ML(G₃₀), ML(C₃₂) and ML(U₃₀) were prepared as described in Åström, J. etal. (1991), as above. The RNA substrates were labeled either overall oron their homopolymeric tails by incorporating radioactively labeledmononucleotides during the in-vitro transcription (see Åström, J.(1991), above, Åström, J. et al. (1992), above). The specificradioactivities of the incorporated radioactive mononucleotides were 40Ci/mmol in the case of the transcription mixture for the overalllabeling or 4 Ci/mmol in the case of the transcription mixture for thetail labeling. Transcribed RNA was purified as described by Moore, C. L.& Sharp, P. A. (1985) Cell, 41, 845-855.

Example 10

In-vitro Deadenylation

The test conditions for the in-vitro deadenylation were as follows: 1 mMMgCl₂, 2.5% (w/v) poly(vinyl alcohol) (Sigma P-8136, Mw 10,000), 100 mMKCl, 0.15 Unit RNAguard, from 5 to 20 fmol of RNA substrate, from 20 to48% (v/v) buffer (20 mM HEPES (KOH), 0.2 mM EDTA, 0.5 mM DTT, 25%glycerol, pH 7) and the appropriate protein fraction (Åström, J. et al.(1992), see above). The reaction volume was 15 or 25 μl and theincubations were carried out at 30° C. The reactions were thenterminated and RNA was purified as described in Moore, C. L. & Sharpe,P. A. (1985), see above. The RNA was analyzed by electrophoresis in 10%polyacrylamide (19:1 acrylamide/bisacrylamide)—7 M urea gels, followedby autoradiography of the resulting gels.

Example 11

2D-thin Layer Chromatography

The two-dimensional (2D thin layer chromatography) was carried out onPEI-Cellulose F plates (Merck No. 5579) in accordance with the method ofKonarska, M. M. et al. (1985) Nature 313 (6003), 552-557. The productsreleased in the deadenylation reactions were analyzed by two-dimensionalthin layer chromatography (2D TLC) carried out in a chamber. The firstmobile solvent (I) was isobutyric acid/concentrated NH₄OH/H₂O;577/38/385 (v/v), and the second mobile solvent (II) was saturated(NH₄)₂SO₄/1 M sodium acetate/isopropanol; 80/18/12 (v/v). After thefirst fractionation, the plates were dried at room temperature for 8hours in order to evaporate off the isobutyric acid. 5′-AMP (SigmaA-1752), 2′-AMP (Sigma A-9396) and 3′-AMP (Sigma A-0386) were used asmarkers. The positions of the markers were determined using UV light(mineral light lamp UVG-54, Ultra Violet Prod. Inc.). The radioactivemolecules were determined by subjecting the resulting PEI-Cellulose Fplate to autoradiography.

Example 12

1D TLC and Quantification of the Nuclease Activity

The deadenylation activity was quantified as follows: L3(A₃₀) RNAsubstrate, labeled with [α³²P]ATP, was incubated in an in-vitrodeadenylation reaction. The reaction products were analyzed by 1D TLCusing mobile solvent III (0.75 M K₂HPO₄, pH 3.5 (H₃PO₄)). The resultingPEI-Cellulose F plate was dried and scanned in a 400 S Phosphorimager(Molecular Dynamics). The fractions containing released [α³²P]AMP weredetermined. The quantity, in mol, of released AMP was calculated on thebasis of the known specific activity of [α³²P]AMP in the RNA substrate.One unit of deadenylation activity corresponds to the release of 1 pmolof AMP/minute.

Example 13

Conditions for the in-vitro Deadenylation

The conditions for measuring poly(A) deadenylation activity inaccordance with the above-described deadenylation test were determinedusing Poly(A) Sepharose fractions as the enzyme source and poly(A)tail-labeled L3(A₃₀) RNA as the substrate. The activity was measured interms of the release of the mononucleotides in the 1D TLC test (seeabove). The requirement of monovalent (potassium and sodium) anddivalent (magnesium, manganese, zinc and calcium) ions for this wasinvestigated.

It was found that divalent cations are not required for thedeadenylation activity and that the optimum monovalent cationconcentration is approx. 100 mM. It was also found that the activity ishigher in the presence of potassium ions than in the presence of sodiumions. It was also found that spermidine had no influence on theactivity.

It was furthermore found that magnesium ions are the preferred divalention and that the optimum concentration is 1 mM. It was furthermore foundthat manganese, zinc and calcium ions can replace magnesium ions to acertain degree. In the presence of 1 mM Mn²⁺, Zn²⁺ and Ca²⁺, theactivity decreases by 17%, 2% and 0.7%, respectively, as compared withthe activity in the presence of Mg²⁺.

Investigations carried out with regard to the pH dependency showed thatthe activity is highest at approx. pH 7.

Example 14

Substrate Specificity

The RNA substrate specificity of the activity was investigated in twoexperiments. In a first experiment, the RNA substrates L3(A₃₀),ML45(U₃₀), ML40(C₃₂) and ML43(G₃₀), whose homopolymeric tails had beenradioactively labeled, were incubated in an in-vitro deadenylation test(see above). The release of radioactive mononucleotides was measured inthe 1D TLC test (see above). The measurement results were used tocalculate the Km and Vmax values for each RNA substrate using theLineweaver-Burke plot (see Table II). In a second experiment, thesubstrate specificity was measured by incubating RNA substratescontaining internal poly(A) segments, followed by plasmid-encoded RNAsequences of increasing length, with Poly(A) Sepharose fractions in anin-vitro deadenylation test. It was found that the RNA substratesL3(A₃₀)X₁₅, L3(A₃₀)X₄₉ and L3(A₃₀)X₁₆₄ were completely resistant todegradation, in contrast to the RNA substrate L3(A₃₀) (see FIG. 3). Itfollows from this that RNA tails composed of adenosine residues are thepreferred substrate and that only 3′ end-located poly(A) tails areeffectively degraded.

Example 15

Determining the Mononucleotide which is Released

The mononucleotide reaction product which is released was determined bymeans of 2D TLC. For this, the Poly(A) Sepharose fraction was incubated,in an in-vitro deadenylation test, with L3(A₃₀) RNA substrate which hadbeen labeled with [α³²P]AMP during the in-vitro transcription. After 20minutes, the reaction products were analyzed by 2D TLC using unlabeled2′-AMP, 3′-AMP and 5′-AMP as markers. This showed (see FIG. 4) that5′-AMP is released during the in-vitro deadenylation reaction enzyme.

Example 16

Kinetics of the Nuclease Activity

In order to determine whether the nuclease activity degrades RNAsubstrates in a progressive or distributive manner, the kinetics wasfirst of all determined in an in-vitro deadenylation reaction in thepresence of 0.5 μl of a Poly(A) Sepharose fraction and x fmol of L3(A₃₀)RNA substrate which had been labeled overall. FIG. 5A shows thatcompletely degraded RNA accumulates before the entire RNA substrate hasreacted. It follows from this that, under these conditions, thedeadenylation reaction was completely finished after 20 minutes. Inanother reaction, the incubation was carried out for 20 minutes in thepresence of an increasing quantity of poly(A). FIG. 5B shows that addingpoly(A) inhibits the reaction and that both non-deadenylated andcompletely deadenylated RNA substrates were present in association withthe highest measured concentration of added poly(A). In a thirdexperiment, the quantity of nuclease activity was altered. Theincubation time was 20 minutes. FIG. 5C shows that both non-deadenylatedand deadenylated RNA substrates were present even in association withthe smallest quantity of added nuclease activity. It follows from thisthat the exonuclease activity is highly progressive, i.e. the enzymedoes not dissociate from the substrate until the adenosine tail has beencompletely degraded.

Example 17

Refolding Experiment (FIG. 6)

The refolding experiments were carried out as described by Dayle et al.(see above) apart from the fact that the refolding took place on the gelovernight at 4 degrees centigrade. The active fractions on the Poly(A)Sepharose chromatography (Ex. 3) were used for these experiments.

What is claimed is:
 1. A process for isolating a poly(A)-specific 3′-exonuclease comprising the following: (a) preparing a crude extract from animal or human cells; (b) precipitating protein present in the crude extract to obtain a precipitate; (c) subjecting the precipitate to chromatography on a weakly basic anion exchanger to obtain a first active fraction; (d) subjecting the first active fraction to affinity chromatography on heparin to obtain a second active fraction; (e) subjecting the second active fraction to chromatography on a strongly basic anion exchanger to obtain a third active fraction; (f) subjecting the third active fraction to affinity chromatography on a dye to obtain a fourth active fraction; (g) subjecting the fourth active fraction to poly(A)-affinity chromatography to obtain a fifth active fraction; and (h) subjecting the fifth active fraction to chromatography on a basic anion exchanger; wherein a poly(A)-specific 3′-exonuclease is isolated.
 2. The process of claim 1, wherein the animal or human cells are thymus cells.
 3. The process of claim 1, wherein the precipitation according to step (b) occurs in ammonium sulfate at approximately 45% saturation.
 4. The process of claim 3, wherein the precipitation according to step (b) comprises the following: (1) precipitating protein present in the crude extract in ammonium sulfate at approximately 25% saturation, then (2) adding further ammonium sulfate such that approximately 45% saturation of ammonium sulfate is present in a supernatant.
 5. The process of claim 1, wherein (1) the precipitate is obtained by precipitation using ammonium sulfate; (2) the first active fraction is obtained using chromatography on DEAE; (3) the third active fraction is obtained using chromatography on Mono Q; (4) the fourth active fraction is obtained using affinity chromatography on a blue dye; and (5) the fifth active fraction is subjected to chromatography on a strongly basic anion exchanger.
 6. The process of claim 5, wherein (1) the fourth active fraction is obtained using chromatography on Blue Sepharose; and (2) the fifth active fraction is subjected to chromatography on Mono Q.
 7. The process of claim 1, wherein (1) the first active fraction is eluted at an approximate 0.17 M concentration of a salt; (2) the second active fraction is eluted at an approximate 1.0 M concentration of a salt; (3) the third active fraction is eluted at an approximate 10% concentration of a 1.0 M concentration of a salt; (4) the fourth active fraction is eluted at an approximate 1.0 M concentration of a salt; or (5) the fifth active fraction is eluted at an approximate 0.35-0.55 M concentration of a salt.
 8. A process for isolating a poly(A)-specific 3′-endonuclease comprising the following: (a) preparing a crude extract from animal or human cells; (b) precipitating protein present in the crude extract to obtain a precipitate; (c) subjecting the precipitate to chromatography on a weakly basic anion exchanger to obtain a first active fraction; (d) subjecting the first active fraction to affinity chromatography on heparin to obtain a second active fraction; (e) subjecting the second active fraction to chromatography on a strongly basic anion exchanger to obtain a third active fraction; (f) subjecting the third active fraction to affinity chromatography on a dye to obtain a fourth active fraction; (g) subjecting the fourth active fraction to poly(A)-affinity chromatography to a obtain a fifth active fraction; (h) subjecting the fifth active fraction to gel filtration to obtain a sixth active fraction; and (i) subjecting the sixth active fraction to chromatography on a basic anion exchanger, wherein a poly(A)-specific 3′-exonuclease is isolated.
 9. The process of claim 8, wherein the animal or human cells are thymus cells.
 10. The process of claim 8, wherein (1) the precipitate is obtained by precipitation using ammonium sulfate; (2) the first active fraction is obtained using chromatography on DEAE; (3) the third active fraction is obtained using chromatography on Mono Q; (4) the fourth active fraction is obtained using chromatography on a blue dye; and (5) the sixth active fraction is subjected to chromatography on a strongly basic anion exchanger.
 11. The process of claim 10, wherein (1) the fourth active fraction is obtained using chromatography on Blue Sepharose; and (2) the sixth active fraction is subjected to chromatography on Mono Q.
 12. The process of claim 8, wherein the precipitation according to step (b) occurs in ammonium sulfate at approximately 45% saturation.
 13. The process of claim 12, wherein (1) the first active fraction is eluted at an approximate 0.17 M concentration of (2) the second active fraction is eluted at an approximate 1.0 M concentration of a salt; (3) the third active fraction is eluted at an approximate 10% concentration of a 1.0 M concentration of a salt; (4) the fourth active fraction is eluted at an approximate 1.0 M concentration of a salt; (5) the fifth active fraction is eluted at an approximate 0.35-0.55 M concentration of a salt; or (6) the sixth active fraction is subjected to chromatography on a basic anion exchanger using approximate 0.17 M concentration of a salt.
 14. A process for isolating a poly(A)-specific 3′-endonuclease comprising the following: (a) preparing a crude extract from animal or human cells; (b) precipitating protein present in the crude extract to obtain a precipitate; (c) subjecting the precipitate to chromatography on a weakly basic anion exchanger to obtain a first active fraction; (d) subjecting the first active fraction to affinity chromatography on heparin to obtain a second active fraction; (e) subjecting the second active fraction to chromatography on a strongly basic anion exchanger to obtain a third active fraction; (f) subjecting the third active fraction to affinity chromatography on a dye to obtain a fourth active fraction; (g) subjecting the fourth active fraction to two rounds of affinity chromatography to obtain a fifth active fraction; (h) subjecting the fifth active fraction to poly(A)-affinity chromatography to obtain a sixth active fraction; and (i) subjecting the sixth active fraction to chromatography on a basic anion exchanger, wherein a poly(A)-specific 3′-exonuclease is isolated.
 15. The process of claim 14, wherein the animal or human cells are thymus cells.
 16. The process of claim 14, wherein (1) the precipitate is obtained by precipitation using ammonium sulfate; (2) the first active fraction is obtained using chromatography on DEAE; (3) the third active fraction is obtained using chromatography on Mono Q; (4) the fourth active fraction is obtained using chromatography on a blue dye; (5) the fifth active fraction is obtained using ssDNA Agarose following by 5′ AMP chromatography; and (6) the sixth active fraction is subjected to chromatography on a strongly basic anion exchanger.
 17. The process of claim 16, wherein (1) the fourth active fraction is obtained using chromatography on Blue Sepharose; and (2) the sixth active fraction is subjected to chromatography on Mono Q.
 18. The process of claim 14, wherein the precipitation according to step (b) occurs in ammonium sulfate at approximately 45% saturation.
 19. The process of claim 18, wherein (1) the first active fraction is eluted at an approximate 0.17 M concentration of a salt; (2) the second active fraction is eluted at an approximate 1.0 M concentration of a salt; (3) the third active fraction is eluted at an approximate 10% concentration of a 1.0 M concentration of a salt; (4) the fourth active fraction is eluted at an approximate 1.0 M concentration of a salt; (5) the fifth active fraction is eluted at an approximate 2.0 M concentration of a salt; (6) the sixth active fraction is eluted at an approximate 0.35-0.55 M concentration of a salt; or (7) the sixth active fraction is subjected to chromatography on a basic anion exchanger using an approximate 0.17 M concentration of a salt.
 20. A composition comprising the poly(A)-specific 3′-exonuclease isolated by the process of claim
 1. 21. A formulation comprising the composition of claim 20 and a pharmaceutically acceptable additive and/or adjuvant.
 22. A process comprising combining the composition of claim 20 and a pharmaceutically acceptable adjuvant and/or additive.
 23. A composition comprising a poly(A) specific 3′-exonuclease, obtainable from calf thymus, that interacts strongly with Heparin Sepharose, catalyses degradation of poly(A)nucleotides at the 3′ end of mRNAs in a processive manner, has a molecular weight of 50 kDa under denaturing conditions and a molecular weight of 180-220 kDa under native conditions, is active in the presence of 1 mM Mn²⁺ ions and is not stimulated by spermidine. 